Analysis of the structure of dissolved marine humic substances and their phytoplanktonic precursors by 1H and 13C nuclear magnetic resonance

Chemical Geology, 40 (1983) 187--201

187

Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands

ANALYSIS OF THE STRUCTURE OF DISSOLVED MARINE HUMIC SUBSTANCES AND THEIR PHYTOPLANKTONIC PRECURSORS BY IH AND 13C NUCLEAR MAGNETIC RESONANCE

MICHAEL A. W I LS O N " * , ANDREW H. GILLAM 2 and PHILIP J. COLLIN 3

~Lincoln College, Canterbury (New Zealand) 2Department of Oceanography, University of British Columbia, Vancouver, B.C. V6T 1 WS (Canada) 3CSIRO, Division of Fossil Fuels, North Ryde, N.S.W. 2113 (Australia) (Received August 5, 1982; accepted for publication January 26, 1983)

ABSTRACT Wilson, M.A., Gillam, A.H. and Collin, P.J., 1983. Analysis of the structure of dissolved marine humic substances and their phytoplanktonic precursors by 1H and ~3C nuclear magnetic resonance. Chem. Geol., 40: 187--201. The structure of dissolved marine humic material and the intracellular and extracellular material from the diatom Phacodactylum tricornutum has been investigated by IH- and 13C-NMR spectroscopy. The results show that carbohydrates, highly-branched alkyl chains and to a lesser extent aromatic materials are important contributors to the structure o f marine humic substances and aqueous extracts of P. tricornutum. There is a close relationship between the chemical structure of P. tricornutum exudate and dissolved marine humic material.

INTRODUCTION

The mass of organic carbon on the earth's surface in the form o f humic substances is approximately five times the organic material in the form of biomass, yet our knowledge of the structure of these complex macromolecules\has been limited b y a lack of suitable analytical tools and, sadly, by a lack o~ interest among organic chemists. A number of independent approaches have been taken to elucidate the structure of humic substances. These include degradative methods such as oxidation and reduction, pyrolysis and conventional analytical methods such as functional group and elemental analysis. All have been useful to varying degrees (Schnitzer and Khan, 1978). The increased sensitivity of Fourier transform methods over continuous *Present address: CSIRO, Division of Fossil Fuels, North Ryde, N.S.W. 2113 (Australia)

0009-2541/83/$03.00

© 1983 Elsevier Science Publishers B.V.

188 wave methods has made NMR a powerful technique for characterising these materials and more recently the solid-state cross-potarisation technique with magic angle sample spinning (CP--MASS) has greatly improved the quality of spectra of humic substances (Hatcher et al., 1980a, b, 1981). Intimate details of the structure of humic substances are not known. It has long been recognised that humic substances of terrestrial origin may be formed from the microbiological and chemical degradation products of lignin, which may react with proteins and amino acids in soils. Another possible reaction is the condensation of sugars with amino acids. Recently, polymethylene and long alkyl chains have been recognised as components of humic extracts (Wilson, 1981). A similarity between humic material and polymaleic acid has also been observed (Anderson and Russell, 1976). Our knowledge of the chemical structure of marine humic materials, in both sedimentary environments and in seawaters is even more limited. In marine systems it is believed that condensation products of sugars and amino acids are important contributors to humic substances. Recently, Hatcher and coworkers have been able to show that paraffinic structures in marine humic acids are also present, and that they can be highly branched (Hatcher et al., 1980). Ishiwatari (1973) has suggested cyclic aliphatic structures may be important contributors to structure. It is clear that the nature of humic materials can vary from source to source, and hence it is pertinent to ask what factors may cause this variability. Firstly, the nature of the parent material may be important since, if the decomposing plant life contains a larger proportion of one class of chemical compounds than another, then the organic matter will contain different proportions of degradation products. Secondly, the degree of microbiological activity will influence the transformation of plant and animal debris which, in turn, will mean that the contribution of weakly and strongly modified organic material to the total will differ in environments with different microbial populations. Of course, climate will also be important since it will influence the nature and density of microbiological and macrobiological life. It should also be borne in mind that if there is a means of transporting humic material from one environment to another, then the humic material in a particular environment may not be that derived from plant, animal and microbiological activity in that environment. Thus the humic material in rivers and lakes may reflect the plant life in the surrounding terrestrial environment (Wilson et al., 1981). Possibly the simplest system in which to investigate the manner by which humification occurs is to study the relationship between dissolved marine humic material in seawater and the major contributor to that humic material, the phytoplankton. In this work the structure of intracellular and extracellular material from the marine diatom Phaeodactylum tricornutum Bohlin is investigated by NMR spectroscopy. Dissolved marine humic substances were also isolated from a large volume (860 1) of coastal seawater to enable comparison to be made by NMR between the naturally occurring dissolved

189

organic matter and that isolated from P. tricornutum. The results presented here extend those o f a preliminary account (Gillam and Wilson, 1983}. MA T ER I ALS AND METHODS

Coastal seawater humic substances The large volume (860 1) of coastal seawater sample was collected from the submerged inlet of the seawater pumping system of the West Vancouver Laboratory, Department of Fisheries and Oceans, Canada, and was stored in 200-1 polyethylene barrels at 18°C prior to filtration. Details of the analytical scheme used for the concentration of marine humic substances are similar to those given by Wilson et al. (1981}. Briefly, the seawater was filtered (GF/C~), acidified to pH 2.0 and passed through columns (30 × 2 cm) of acetone-extracted Amberlite XAD-2 ® resin. After the passage of the sample, the column was rinsed with cold distilled water until the eluate was chloridefree. The humic substances were then eluted by the passage of t>5 bed volumes o f a 1 : 1 (v/v} mixture of methanol and 2 M ammonium hydroxide. The solution was evaporated to dryness in a rotary evaporator at < 35°C. The dried humic extract was then redissolved in 100 ml of distilled water and exhaustively extracted with chloroform to remove lipoidal material. The aqueous solution was filtered (Whatman ® No. 1) and then rotary evaporated at <35°C. The dried humic extract was stored in vacuo over Mg(CIO4)2. Chemical analysis showed 43.6% C and 5.74% H. Organic extracts o f Phaeodactylum tricornutum A unialgal axenic culture of Phaeodactylum tricornutum Bohlin was obtained from the Pacific Northeast Culture Collection at the University of British Columbia. Initially, 1-1 axenic cultures of P. tricornutum were grown in charcoal-treated seawater, which had been autoclaved prior to the addition of sterile ES enrichment. Cells were grown to late exponential phase on a 16 : 8 (light:dark) cycle at 18°C under 60 tiE m -~ s -1 illumination. A largescale unialgal culture was subsequently grown in a 200-1 polyethylene drum, using charcoal-treated filtered (0.45 um) seawater containing ES enrichment, minus EDTA and vitamins. Cells were grown to late stationary phase and then removed via continuous centrifugation (Sharpies ® TL). The filtrate was acidified (pH 2.0) and passed through XAD-2®resin in a similar manner as that described for the coastal seawater sample. Both the aqueous and chloroform-soluble materials obtained from the extracellular products o f P. tricornutum were dried and stored in vacuo over Mg(CIO4}2. Chemical analysis o f the water-soluble material showed 45.5% C and 5.95% H. The cellular material, 84.6 g (wet weight) was placed in a precleaned (Soxhlet extraction, CHCI3, 24 hr.) Pyrex ® extraction thimble and was extracted continuously with CHC13 (7 days, with fresh solvent each day). The

190 chloroform-soluble material was filtered (Whatman ® No. 1), dried over Na2SO4 and r o ta r y evaporated at <35°C to an oily residue. The material was stored in vacuo over Mg(C104)=. The chloroform-extracted residue was dried to remove residual chloroform and then extracted for 48 hr., under nitrogen, with 500 ml o f 0.5 M NaOH. The cellular debris was removed (Whatman ® No. 1) and the alkaline solution was passed over a 15 X 1 cm column of Dowex ® 50W-X8 (H+ form). The neutral extract was acidified to pH 2.0 and passed (5X) over a 15 × 1-cm column of precleaned XAD-2 ® resin. The column was rinsed with cold distilled water and the adsorbed organic material was eluted with 300 ml of a 1 : 1 (v/v) m i xt ure of MeOH--2 M NH4OH. The humic ex tr ac t was rotary-evaporated at <35°C and the dried residue was stored in vacuo over Mg(C104)2. Chemical analysis showed 43.0% C and 6.13% H. NMR SPECTRA 1H- and ~3C-NMR solution spectra were determined on a Jeol ® FX90Q spectrometer. The sample (~20~mg) was added to 0.5-cm 3 deuterium oxide. A few drops o f 1 mol dm -3 sodium d e u t e r o x i d e were added until the sample dissolved. 1H-spectra were obtained at 89.99 MHz under hom ogat ed decoupling conditions in which the HOD peak produced by adventitious water impurities and p r o t o n exchange was irradiated. The radiofrequency (RF) level of the HOD irradiation was optimised for each sample. That is, sufficient RF level to reduce the HOD peak to zero, but n o t enough to distort the intensity of the humic material in the vicinity of the HOD peak. Spectra were determined using a 45 ° pulse and 8K data were acquired using 1500-Hz spectral width. Acquisition time was 2.727 s, with a pulse delay of 2.0 s. Approximately 1000 scans were collected for adequate signal to noise ratio. Chemical shifts are q u o t e d with respect to internal tetramethylsilane (TMS) but were measured with respect to external TMS. A capillary inserted into the sample tube was used as reference. The values so obtained were corrected by measuring the chemical shifts of n-butanoic acid with respect to both internal and external TMS. Solution 13C-NMR spectra were determined at 22.5 MHz. 8K data were collected using a 7000-Hz spectral width. Acquisition time was 0.584 s. A pulse delay of 1.0 or 5.0 s were used with, and without, inverse gated decoupling. Up to 60,000 scans were collected. 13C-CP--MASS spectra were obtained on a Bruker ® CXP-100 instrument. A r o t o r consisting of a barrel of b o r o n nitride and a base of Kel-F ® was used. R o t o r speed was ~ 3 . 8 kHz. Recycle time was varied from 0.3 to 1 s. A variety of co n tact times from 0.5 to 3 s were employed. T he H art m ann--H ahn condition was set using a sample of he xa met hyl benzene. Chemical shifts were measured with respect to external h e x a m e t h y l b e n z e n e (by storing the h e x a m e t h y l b e n z e n e spectrum in another c o m p u t e r m e m o r y block) but are

191

quoted with respect to TMS. It is assumed that the chemical shifts of hexamethylbenzene with respect to TMS are the same in solution as in the solid state. RESULTS AND DISCUSSION

Assignments from 'H-NMR spectra The 1H-NMR spectra (Fig. 1) can be broadly divided into four regions: 0.5--1.8 ~ (alkyl protons attached to carbons removed from aromatic rings or carboxylic groups, H0); 1.8--3.0 8 (largely protons attached to carbon a, to aromatic rings and carboxylic groups, Ha); 3.0 4.75 ~ (alcohol and ether protons attached to carbon a--to oxygen, HR_ O and 6.4--9.0 ~ (largely aromatic and olefinic protons, HAr ). The approximate proportions of these hydrogen types (excluding exchangeable hydrogens) are shown in Table I. Clearly, the proportion of hydrogen which is aromatic in all three extracts is small (5--7%). Most of the IH-NMR signal arises from aliphatic protons. A moderate proportion of the aliphatic protons are attached to ether or alcohol carbons (HR_o). These protons are present in greater proportion in the intracellular extract of P. tricornutum than in either the extracellular extract or the seawater humic material. The proportions of all hydrogen types are very similar in the seawater humic substance and in the extracellular extract (Table I). The two extracts also appear to have similar fine structure in their spectra. There appears to be

[

•

]

•

_

_ .

~-

, 4 .

c I

I

10

i

l

i

I

i

5

I

i

i

0

~) (ppm) Fig. 1. I H - N M R

spectra of: (a) seawater bumic substances; (b) extracellular material from

P. tricornutum ;and (c) intracellular material from P. tricornutum.

t~

TABLE I A p p r o x i m a t e estimates o f c a r b o n t y p e s in m a r i n e s a m p l e s % Carbon types

Coastal seawater P. t r i c o r n u t u m

% Hydrogen types

m e t h o d *~

carboxyl*:

ary1.3

acetal O-alkyl or ketal

alkyl

method

aryl HAt

O-alkyl HR__O

Ha (.4)

Ho(*S)

a b a

14 15 15

18 21 19

2 3 3

18 18 15

49 43 49

b

7

19

32

42

b

5

22

27

46

a

21

6

7

42

25

b

6

30

26

37

extraceUular material P. t r i c o r n u t u m

intracellular material * 1 M e t h o d : a = by CP--MASS N M R ; b = b y s o l u t i o n F T NMR.

*2Includes salt ester a n d a m i d e c a r b o n . * 3Includes olefinic c a r b o n . **Largely h y d r o g e n a t t a c h e d t o c a r b o n a t o a r o m a t i c rings or e l e c t r o n w i t h d r a w i n g groups such as c a r b o x y l . * 5Mainly alkyl p r o t o n s ;~ or f u r t h e r f r o m a r o m a t i c rings or e l e c t r o n w i t h d r a w i n g groups.

193

at least three singlet resonances at 8.4, 3.3 and 1.85 5 in both spectra. The resonance at 8.4 8 possibly arises from formate ion. The extracellular extract has an additional resonance at 1.8 8, and the seawater extract an additional resonance at 3.63 8. The two resonances at 1.21 and 0.85 5 appear to be well resolved in the extracellular material of P. tricornutum but are poorly resolved in the spectrum of the seawater humic sample. The IH-NMR spectrum of the intracellular extract differs quite considerably from that of the extracellular extract or the seawater sample. The alkyl region 0.5--1.8 8 contains two broad resonances at 0.95 and 1.37 5. The resonances centred around 2.0 8 are n o t so prominent as in the other spectra and are resolved into two regions centred at 2.08 and 2.37 8. An additional resonance at 2.76 8 is present. The R--O region contains a prominent resonance at 3.8 5 which dwarfs the other resonances in this region. In the aromatic region a strong signal at 7.35 8 is also present.

Assignments from 13C-NMR spectra The 13C-NMR spectra shown in Fig. 2 were determined by CP--MASS NMR. The 13C-spectrum of the seawater humic material was also determined by solution 13C-NMR. Like the 'H-spectra, several broad ranges of resonance

(b)

(c)

[ppm}

Fig. 2.13C-CP--MASS spectra of: (a) seawater humic substances', (b) extraceilular material from P. t r i c o r n u t u m ; and (c) intraceUular material from P. t r i c o r n u t u m .

194 can be recognised in all the 13C-NMR spectra. These have chemical shifts (with assignments in brackets:) 190--160 5 (COOH, ester, salt and amide), 110--160 5 (aromatic and olefinic carbon), 95--110 5 (dioxygenated carbon, e.g. acetal), 64--95 5 (oxygenated alkyl carbon of ethers and alcohols), 0--64 5 (alkyl carbon). The alkyl resonance is centred ~ 3 0 ppm which indicates significant quantities of (CH2)n in the extracts. Apart from the broad envelopes there appears to be some additional resolution of carbon types in the O-alkyl and alkyl regions. The extra-cellular material has a shoulder at 15--24 ppm due to carbons a, ~ and ~/ from the end of alkyl chains (Fig. 2b). There are also shoulders at 38 and 46 ppm in the spectrum of the extracellular material which probably arises from carbon a - t o aromatic rings or a--to carboxyl structures. These shoulders are more clear cut in the spectrum of the intracellular material (Fig. 2c). The resonance at 46 ppm is also present in the spectrum of the seawater humic sample (Fig. 2a). Resonances are also present in the three spectra at 56 ppm. They are evident as a clear cut peak in the spectrum of the intracellular material (Fig. 2c) but are only part of the overall broad aliphatic band in the spectra of the other extracts. The 56-ppm resonance arises from--CH20~carbon in ethers and alcohols.

Quantitative determination of carbon types There have been two approaches to the quantitative determination of carbon types in complex mixtures by NMR. Neither approach is straightforward (Hatcher et al., 1980a, b; Newman et al., 1980; Wilson et al., 1981). In the first, and more established approach, the sample is dissolved in a suitable organic solvent and the 13C-NMR spectrum determined, leaving sufficient delay between pulses to ensure that complete relaxation occurs for all carbons in the sample. This is usually 3--5 times the longest TI present in the sample. In addition, since nuclear Overhauser enhancements (NOE) can occur because of proton decoupling, the decoupler is gated off between pulses (inverse gated decoupling). For humic substances 0.5 • 105--10 s transients are needed to obtain a spectrum with a reasonable signal to noise ratio, hence the use of long pulse delays between successive transients is prohibitive. Relaxation behaviour of freshwater aquatic and terrestrial humic substances have been studied and for complete relaxation a pulse delay of at least 5 s is needed. Nevertheless, estimates of aromaticity (the fraction of carbon which is aromatic) show little variation with pulse delay, which indicates that relaxation times of carbon atoms in the sample are similar. Inverse gated decoupling experiments revealed small and similar NOE's for aromatic and aliphatic carbon, but for accurate estimates of carboxyl carbon it was necessary to gate off the decoupler between pulses and increase the pulse delay. In summary, the two studies of relaxation in solution reported so far show that quantitative estimations of carbon types can be made from 13C-

195

NMR spectra o f humic substances, Nevertheless, a preliminary investigation of relaxation behaviour is necessary to confirm similar behaviour between different humic acids. Fig. 3 shows the effect of varying pulse delay, and gating the decoupler o f f b e t w e e n pulses while determining the solution 13C-NMR spectrum of the marine humic material investigated in this work. Clearly, very short delays similar to those used on other humic samples have little effect on the measured aromaticity. Increasing the pulse delay from 1 to 5 s increases the apparent aromaticity from 0.14 to 0.18, which is only just larger than experimental error. However, it is quite clear that if a subdivision of aliphatic types into alkyl, O-alkyl, acetal and carboxyl is to be undertaken, increasing the pulse delay from 1 to 5 s has a significant effect on relative signal intensities. For example, the fraction of carbon classified as alkyl decreases from 0.55 to 0.46. There is also a small nuclear Overhauser enhancement at a pulse delay o f 5 s. When the decoupler is gated o f f between pulses the apparent aromaticity of the humic material increase from 0.18 to 0.22. The second m e t h o d of measuring the aromaticity of humic substances involves cross-polarisation 13C-NMR with magic angle sample spinning (CP-MASS). This is a relatively new technique which has become invaluable in the analysis of insoluble materials such as polymers and coals. Only recently

'sf

50 ~5 ~0

ALKYL I

25

--o o

O--

o~ 20

R-O

15 I

I

~' C C

25

20 15

-~ lO 15

F

1°I

I

I

I

o ~o O-

ARYL I

I

I

I

I

o --o O--

COOH

5

°E 5

I

I

I

I

• L 1

I

Q I 2 Pulse

I I 3 ~ delay /s

I 5

.~0

R,.. 0

Fig. 3. E f f e c t o f p u l s e d e l a y o n relative signal i n t e n s i t i e s • = c o m p l e t e d e c o u p l i n g ; o = gated decoupling.

196 has it been applied to the analysis of humic materials and soils. The advantages of CP--MASS for quantitative measurements are: (1) no nuclear Overhauser enhancement; (2) a signal enhancement of up to four; (3) rapid relaxation; and (4) no solubility limits. Nevertheless, there are several inherent assumptions in quantitative analysis by CP--MASS which have, as yet, not been fully justified. In the CP--MASS process polarisation of the carbons is brought about by matching the energy levels of carbons and protons. This is done by altering the magnetic field seen by the protons by means of the spin locking technique in which after applying the initial 90 ° pulse the phase of the applied radiation is changed so that it is collinear with the magnetisation vector in the rotating frame. When the energy levels of carbon and proton are matched, then the population differences between carbon and hydrogen are equalised by energy transfer. This energy transfer takes a finite time, the cross-polarisation time. Since the cross-polarisation time is dependent on the proximity of carbons to protons it is possible that some nonprotonated carbons are n o t fully polarised or worse, not polarised at all. Also if different carbons relax at different rates, then at a given contact time, the polarisation of all carbon types may not be the same and this can lead to erroneous estimations. On top of all this, it is necessary to optimise the recycle time between pulses. Too short recycle times may result in incomplete relaxation. Too long recycle times result in inefficient use of spectrometer time. This is particularly critical with humic substances for which large numbers of transients are needed (~2.104). The effects of varying recycle times and cross-polarisation times have been studied for coal samples which are closely related to humic substances. A contact time o f / > 1 ms is necessary to ensure maximum cross-polarisation to carbon atoms. In general, aromatic carbons cross-polarise much more slowly, so that at very short contact times the aromaticity may be underestimated (Van der Hart and Retkofsky, 1976). Two other types of experiments have been performed to demonstrate the quantitative nature of CP data. Van der Hart and Retkofsky have found that the measured aromaticity of coal extracts at optimum contact or recycle times are similar whether determined by solution- or solid-state NMR. These results have been essentially confirmed by our group working on other coalderived products. Experiments have also been performed in which crosspolarisation data have been compared with long-term Bloch decay 13C solidstate experiments. Both types of experiments have indicated similar values for the aromaticity of one American coal (Maciel et al., 1979} but conflicting results have been obtained with others (Dudley and Fyfe, 1982). No studies of the effects of contact time on relative signal intensities have been reported for humic substances, although comparisons between solutionand solid-state spectra have been reported (Hatcher et al., 1980a,b; Newman et al., 1980; Wilson et al., 1981). The effects of contact and recycle time were investigated for the seawater humic material used in this work. Some typical spectra are illustrated in Fig. 4. Short contact times (< 1 ms) significantly re-

197

(b)

~^

2;0

~.

~

/

1;0 (ppm)

Fig. 4. Short contact time (CT) CP--MASS spectra of seawater humic substances.

duced the contribution of aromatic and carboxyl signals, to the spectra. At longer contact times (2 ms) the apparent aromaticity of the seawater sample appeared to be similar when measured by CP--MASS NMR or b y solution NMR (Table I). If anything the aromaticity may be slightly underestimated by CP--MASS.

Structure o f Phaeodactylum tricornutum extracts The cellular carbohydrates of P. tricornutum have been investigated by Ford and Percival (1965). They were able to separate a water-soluble glucan and low-molecular-weight carbohydrates, using mild chlorite and cold alkali treatment. Laminaribiose, glucose, methylinositol, laminaritriose galactosyl (1--3) laminaribiose, myoinositol and scyllitol were identified. From hot alkaline solution t h e y were also able to identify glucuronic acid and a sulphonated glucuronomannan. Figs. 1 and 2 clearly show the presence of large amounts of acetal or ketal carbons and O-alkyl carbons and protons attached to carbons a - - t o oxygen in the intracellular and extracellular extracts from the P. tricornutum culture investigated in this work, thus confirming that large quantities of carbohydrates are present in these extracts. The 13C-NMR spectra of both intraceUular and extraceUular extracts have resonances centred at 30 ppm, which as already noted, suggests the presence of methylene carbon. Nevertheless, the alkyl resonance is broad and extends to ~ 6 0 p p m which suggests that m a n y o f the alkyl chains are branched. This is confirmed b y the 1H-NMR spectra which show very prominent signals at 0.9 ppm due to methyl groups 7- or further from aromatic rings or polar groups. Thus it is clear that a substantial proportion of the alkyl substituents in b o t h extracts are highly branched with many methyl groups.

198

There appears to be substantially more aromatic and/or olefinic carbons in the extracellular extract than the intracellular extract, but the aromatic proton c o n t e n t of both extracts is not large. The possibility exists that some of the phenolic aromatic protons may exchange in NaOD/D20 with deuterons. The peak at 8.4 ppm in the ~H-spectrum possibly arises from formate ion which is often formed from humic acids and related materials. Both intracellular and extracellular extracts contain appreciable proportions of carboxylic carbon (Fig. 2; Table I). Ford and Percival (1965) have shown that glucouronic acids {structure 1) are important components of

C02H

J~--O

H

OH

alkali extracts of the diatom and thus these may be responsible for some of the carboxylic carbon observed in the NMR spectra. Nevertheless, it cannot account for all the carboxylic carbon since structure (1) suggests at least four mono-oxygenated aliphatic carbons for each carboxylic carbon. Table I shows that there is only one (extraeeUular material) or two (intracellular material) mono-oxygenated aliphatic carbons per carboxylic carbon in the two extracts respectively. Some of the carboxylic carbon could be accounted for if it is attached to aromatic rings or olefins, but this cannot be the only explanation for intracellular material, since there is not enough aromatic plus R--O carbon (assuming 1 COOH for 4 R--O) to account for all the carboxylie carbon. Hence, at least a proportion of the earboxylic carbon must be attached to alkyl carbon. Structure o f seawater humic materials

Fig. 1 shows that, in general features the ~H-NMR spectrum of the seawater humic material resembles that of the spectrum of the extracellular extract of P. tricornutum. Fig. 2 shows that the general features of the 13C-spectra of seawater humic material and extracellular extract are also similar. In addition, both seawater and extracellular materials have similar C/H atom ratios: 0.63 and 0.64, respectively. By comparison the C/H atom ratio of the intracellular material is 0.58. Thus it is reasonable to assume that the extracellular extract of P. tricornutum and the seawater humic material have similar proportions of functional groups. Either, the extracellular material from P. tricornutum makes a significant contribution to the seawater humics or other contributing material {e.g., from other diatoms or phytoflagellates} has a similar structure to the P. tricornutum extracellular material. The fact that the humic material resembles the extracellular material more

199

than the intracellular material is not surprising since the abundance of plant remains is controlled b y grazing organism in marine environments. This is in contrast to vegetation in the terrestrial environment where less than 10% of plant material on average is eaten. In the marine environment ~ 9 0 % is metabolised. Thus exudates of diatoms might be expected to play an important part in producing seawater humic material (Menzel, 1976). It is clear that seawater humic material contains appreciable quantities of carbohydrate and most probably these are of a similar t y p e to those present in the extracellular material from P. tricornutum. As already noted, uronic acids are important components of P. tricornutum and thus some of the carboxylic acid groups in humic acids could be from this source. Our results show that aromatic components are present in the extracts of P. tricornutum and also in the marine humic materials. The presence of aromatic precursors of dissolved marine humic substances has been detected by other groups (Craigie and MacLachlan, 1964). Several Phaeophyceae were shown to produce extracellular UV-absorbing materials. Preparative isolation of the exudate o f Fucus vesiculosus indicated these extracellular extracts contained polyphenolic components. In addition, the UV spectrum of an extracellular vesiculosus extract was similar to that reported for water from the Irish Sea (Armstrong and Boalch, 1961). Thus it appears that there is a possible source of aromatic precursors in macrophytic or other marine algae which could account for the presence of aromatic moieties in marine humic substances. Another possible source is the uronic acids. It is clear that in aquatic solution conditions such as pH, temperature, solvent, cations and the nature of the acid strongly influence the transformation and degree of degradation. It is n o t e w o r t h y that aromatic materials such as 3,8-dihydroxy-2-methyl chromone (2), benzene carboxylic acids (3), 2-furoic acid and a series of phenolic c o m p o u n d s (4--7) can be formed from uronic acids at pH 7 (Popoff and Theander, 1976).

H~O-~ v

Me

C02[~ OH

~ ~OH 0 {2)

OH

OH

OH

(5)

(~1

[~ Ac

(s)

(3)

OH

CH3 [~OH

OH

OH (7)

200

The importance of uronic acids as precursors of both marine and terrestrial humic material does not appear to be fully appreciated. Not only are they important components of marine diatoms but they are also constituents of animal microbial and plant polysaccharides. It is worth noting that furoic and phenolic materials are isolated from soil humic materials (Schnitzer and Khan, 1978). Partial structures of humic materials are often formulated as consisting of phenolic structures held together by hydrogen bonds and covalent bonds (Schnitzer and Khan, 1978). It is possible that many of these aromatic and furoic structures derive from uronic acids as well as lignin. A third source of aromatics in marine environments may be a contribution from terrestrial sources. Ishiwatari (1973), Dereppe et al. (1980) and Hatcher et al. (1981) have examined humic structures from marine sediments. All extracts are highly aliphatic but those from coastal environments are more aromatic in nature. Most probably the aromaticity and the carbohydrate content of seawater and sedimentary humic substances will vary from source to source, and will depend on the degree of contribution of different marine vegetations and the relative contribution from terrestrial sources. In summary, our results clearly indicate the similarities in chemical structures of a marine diatom extract and marine humic material. They also show that carbohydrates, highly-branched alkyl chains and to a lesser extent aromatic materials are important constituents of marine humic materials. The important contribution or uronic acids to the structure of marine humic substances is also demonstrated. Clearly, the close relationship between the chemical structure of marine exudate, diatom extrudate and dissolved humic substances must mean that microbiological activity in transforming exudate material into other substances in marine environments must be low.

REFERENCES Anderson, H.A. and Russell, J.D., 1976. Possible relationship between soil fulvic acid and polymaleic acid. Nature (London), 260: 597. Armstrong, F.A.J. and Boalch, G.T., 1961. The ultra-violet absorption of sea water. J. Mar. Biol. Assoc. U.K., 41 : 591--597. Craigie, J.S. and MacLachlan, J., 1964. Excretion of coloured ultraviolet absorbing substances by marine algae. Can. J. Bot., 42: 23--33. Dereppe, J.M., Moreaux, C. and Debyser, Y., 1980. Investigation of marine and terrestrial humic substances by 1H and ~3C nuclear magnetic resonance and infra red spectroscopy. Org. Geochem., 2: 117--124. Dudley, R.L. and Fyfe, C.A., 1982. Evaluation of the quantitative reliability of the ~3C CP/MAS technique for the analysis of coals and related materials. Fuel, 61: 651--657. Ford, C.W. and Percival, E., 1965. The carbohydrates of Phaeodactylum tricornutum, Parts I and II. J. Chem. Soc., pp. 7035--7046. Gillam, A.H. and Wilson, M.A., 1983. Application of 13C-NMR dissolved to the structural elucidation of their phytoplanktonic precursors. In: R.F. Christman and E. Gjessing (Editors), Aquatic and Terrestrial Humic Materials. Ann Arbor Science Publishers, Ann Arbor, Mich., pp. 25--35.

201 Hatcher, P.G., Rowan, R. and Mattingly, M.A., 1980a. IH and 13C NMR of marine humic acids. Org. Geochem., 2: 77--85. Hatcher, P.G., VanderHart, D.L. and Earl, W.L., 1980b. Use of solid state 13C NMR in structural studies of humic acids and humin from Holocene sediments. Org. Geochem., 2: 87--92. Hatcher, P.G., Schnitzer, M., Dennis, L.W. and Maciel, G.E., 1981. Aromaticity of humic substances in soils. Soil Sc. Soc. Am. J., 45: 1089--1093. Ishiwatari, R., 1973. Chemical characterization of fractionated humic acids from lake and marine sediments. Chem. Geol., 12: 113--126. Maciel, G.E., Bartuska, V.J. and Miknis, F.P., 1979. Characterization of organic material in coal by proton decoupled ~3C nuclear magnetic resonance with magic angle spinning. Fuel, 58 : 391--394. Menzel, D.W., 1976. In: E.D. Goldberg (Editor), The Sea, Vol. 5. Wiley, New York, N.Y., pp. 659---678. Newman, R.H., Tate, K.R., Barron, P.F. and Wilson, M.A., 1980. Towards a direct nondestructive method of characterizing soil humic substances using ~3C NMR. J. Soil. Sci., 31: 623--631. Popoff, T. and Theander, O., 1976. Formation of aromatic compounds from carbohydrates. Acta Chem. Scand., 830: 705--710. Schnitzer, M. and Khan, S.U., 1978. Soil Organic Matter. Elsevier, Amsterdam, 319 pp. (see especially pp. 45--47). Van der Hart, D.L. and Retkofsky, H.L., 1976. Estimation of coal aromaticities by proton decoupled carbon-13 magnetic resonance spectra o f whole coals. Fuel, 55: 202--206. Wilson, M.A., 1981. Application of nuclear magnetic resonance spectroscopy to the study of the structure of soil organic matter. J. Soil Sci., 32: 167--186. Wilson, M.A., Barron, P.F. and Gillam, A.H., 1981. The structure of freshwater humic substances as revealed by ~3C NMR spectroscopy. Geochim. Cosmochim. Acta, 45: 1743--1750.